In protein
engineering and design, enhancing the stability of proteins is of
great interest by both basic research and industrial applications.
Disulfide engineering as one of these approaches has become a
powerful experimental modification tool to alter protein stability.
Recent studies suggest that inclusion of structural features in
disulfide engineering would improve the likelihood of a stabilizing
mutation. It is desirable to choose residue pairs that are in
relatively flexible regions close to the protein surface. Here, the
software DIScover has been introduced. The software based on structural status that residues are
located in and aminoacid preferences propose a list of piars for disulfide engineering. Moreover, user
can check each desirable mutations to generate mutant PDB. the
mutant PDB will be minimized by NAMD.

Explanation:

Given the PDB file, the coordination of beta carbons
will be isolated. In the case of glycine residues, according to the
coordinates of the residue backbone atoms the beta carbon location
will be modeled. Cβ_Cβ distance in the all
possible pairs will be calculated. Pairs of residues with distance
less than 4.5 Å will be separated. Both structural parameters and
amino acids preferences for each pair will be evaluated.
Surface accessibility and
normalized B-factor build the structural information criteria
section; stability index, hydropathy scale, amino acid volume,
cavity limitation and steric overlapping have been noted in the
amino acids characteristics section. According
to the criteria of the total score will be calculated in each case. Score 3, 2, 1 for average accessibility, average
normalized B-factor and amino acids preferences have been
considered, respectively. Pairs which have not been satisfied at
least one of these criteria will be scored zero thus predicted as
destabilizing mutations and will be removed. The
remaining pairs will be sorted from most to least according to their
points and will be displayed.
There are two modes for using software; simple and advanced mode.

Inter/Intra-chain:intra-chain
mutations are those pairs that are
in same chain. inter-chain mutation
are mutations in interfacial region
of protein. if all kind
of mutation is regarded it should be
selected as "Any".

Sequential
Distance:Number
of residues exist between two mutant
residues in same chain.

Advanced
mode:

Advanced mode has been implemented to broaden the software
facilities. Most of the options can be manipulated in this section.
Average ACC, average normalized B-factor and change in volume upon
mutation can change to those values that the user wishes to apply.
If the user is demanded to find all potential disulfide bonds
regardless of the stabilizing and destabilizing effects no feature
should be selected in this mode. In addition, the distance criterion
can be changed from the Cβ_Cβ to Cα_Cα
distance.Through
manipulating options, the user can limit the output of the software;
the sequential distance option makes it possible to remove disulfide
bonding with sequential neighbors, while by using the accessibility
option, situation of mutation (as exposed, buried and intermediate)
can be determined.

Distance Criterion:Residue
pairscan
be filtered according to Cb-Cb orCa-Ca
distance.(
default value for Cb-Cb distance: less than 4.5 A and for Ca-Ca
distance: less than 6.5 A)

Arrangement of feature:the
order of features tobe usedwiththefollowing
option to filter desired pairs.

The logical
operator between features:for example if arrangement of features in previous
section is determined as: "change in volume" "surface
accessibility" "temperature factor", and the first logical operator
is selected as "AND" and the second one is selected as "OR", then
the software filter residue pairs with appropriate (change in volume
AND surface accessibility) OR appropriate temperature factor from
those with proper distance criteria. the value of each feature can
be determine too (see next option).

Favorablevalue
of Structural feature:2
text boxes in front of each structural feature have been considered;
make it possible to specify value of each feature.

Sample of
proposed list of residue pairs:

For
each query a chart of several residue pairs will be shown. each row
for one pair; The
first six columns (columns 3-8) include information about first
residue as it's chain, residue number, residue name, secondary
structure, surface accessibility percentage, average normalized
temperature factor (average B-factor of backbone and Cb atoms).The
second six columns(columns
9-14)contains
thesameinformation which
related to the secondresidue.
change in volume upon mutation is shown in the 15th column. the 16th
and 17th columninvolves
thetype
of mutationas
intra/inter-chain and Ca_Ca or Cb_Cb distance between two residue
according to user's selection, respectively.
the last column show the calculated score for respective pair.
Score 3, 2, 1 for average
accessibility, average normalized B-factor and amino acids
preferences have been considered, respectively.

The checkbox has been implemented for each proposed
pairs. The user cancheck
all residue pairs that wish to mutate into cysteine. Click mutate to
apply mutations that are checked and create mutant coordinate file.

Final output:

A:3D
representation of the mutant using Jmol Java plugin. By clicking on
jmol panel user can manipulate the structure as identify residue
names and numbers, drag, rotate and zoom in or out by using scroll button.

D:By
clicking on jmol some options will be displayed. the user can
utilize these option and manipulate 3D structures.

E:The
mutant coordinate can be download from this section. this
coordination file get from NAMD/VMD by minimization over 1000 steps.

Attention:

Due to the minimization with NAMD
if your PDB file contains DNA associated with protein, please
isolate protein from it, then upload your coordinate file,
otherwise, the software will be crashed and will return no mutant
coordinate.

Appendix:

Structural feature calculation methods:

Structural feature

Method

surface accessibility

Accessibility is
defined as the accessible surface area (ASA) of the residue
in native protein divided by the ASA of the residue in an
extended tripeptide Ala-X-Ala conformation. Accessible surface area has been calculated by DSSP (Kabsch and Sander, 1983) software. Extended state
values with the units in
A2are
as fallow: Ala-110.2; Arg-229.0; Asn-146.4; Asp-144.1;
Cys-140.4; Gln-178.6; Glu-174.7; Gly-78.7; His-181.9;
Ile-185.0; Lys-205.7; Leu-183.1; Met-200.1; Pro-141.9;
Phe-200.7; Ser-117.2; Thr-138.7; Trp-240.5; Tyr-213.7;
Val-153.7 (Ahmad, et al., 2004).

Temperature factor

Temperature factor has been calculated as an
average of the temperature factors of backbone and Cβ atoms
from the PDB associated with the native residue. B-factors
were normalized due to compensation of the differences among
structures. Commonly negative values related to rigid region
, between 0.01 and 2 as intermediate and more than 2
considered as high flexible regions
(Sonavane,
et al., 2013; Zhang, et al., 2009). B-factor has been
normalized using the following equation:

Bnorm=
(B-μ)/σ

where B is the
actual B-factor, μ and σ represent the average and the
standard deviation of the backbone and Cβ
B-factor values for the particular PDB structure.

change in volume

Subtraction of
standard Voronoi volume of two cysteine involving in
disulfide bond from the sum of standard Voronoi volumes for
the pair of mutated residues determines the change in
volume. Macromolecular Geometry webpage have been used to
get standard Voronoi volume of residues through the URL,
http://bioinfo.mbb.yale.edu/hyper/mbg/SurfaceVolumes/StdCoreVolumes.xtalatoms(Gerstein, et
al., 1995).

Bioinformatics Research Group
Faculty of Biological Sciences
Tarbiat Modares University